Genotoxicity and mutagenicity evaluation of isoquercitrin-γ-cyclodextrin molecular inclusion complex using Ames test and a combined micronucleus and comet assay in rats

Abstract

Flavonoids such as quercetin and its glucosides, especially isoquercitrin are well known as anti-inflammatory, anti-allergic, and anti-carcinogenic, etc. The safety of isoquercitrin formulations needs to be established prior to their use in functional food applications. The mutagenicity and genotoxicity of the IQC-γCD inclusion complex were assessed with three standard assays of the bacterial reverse mutation assay (Ames test) and using a combined in-vivo micronucleus and comet assay under the Organisation for Economic Co-operation and Development (OECD) guidelines. In combined rat bone marrow micronucleus and rat liver comet assay performed in male Sprague Dawley (SD) rats, the various doses of IQC-γCD inclusion complex (max. 2000 mg/kg bw) and positive controls ethyl methanesulfonate (EMS) and mitomycin C (MMC), respectively, and negative control (vehicle) were administrated. The results of the Salmonella typhimurium mutagenicity assay (strains TA100, TA1535, WP2uvrA, TA98, and TA1537) after exposure to the IQC-γCD inclusion complex with the absence and presence of the metabolic activation system (S9 fraction from rat liver) revealed a weakly positive response but with no biologically relevant mutagenicity at the conditions examined according to recommended regulatory guidelines. The combined micronucleus and comet assay results reveal that the IQC-γCD inclusion complex did not induce in-vivo genotoxic potential or indication of any oxidative DNA damage in rat liver tissues. Altogether, considering the results of the study, it is unlikely that the consumption of IQC-γCD inclusion complex as food or supplement would present any concern for humans regarding the mutagenicity and genotoxicity.

INTRODUCTION

Plant-based ingredients are the backbone of modern nutraceutical preparations and they are major contributors to the health care industry worldwide. Plants harbor a large number of bioactive ingredients (secondary metabolites) invaluable in controlling some diseases at reasonable low cost. Isoquercitrin (a glycosylated form of quercetin) is one of the abundant flavonoids in plant-based foods and the most common antioxidant in the human diet (Valentová et al., 2014; Li et al., 2011) with potential health benefits. Isoquercitrin’s redox behavior is related to its ability to act as a pro-oxidant in certain conditions and is mainly associated with physiological processes involving laccase to generate reactive pro-oxidant intermediates by one-electron oxidation (Moţ et al., 2014; Boots et al., 2003). Quercetin is reported to induce a variety of mutagenic and genotoxic effects in-vitro; however, no specific in-vivo effects are available. Recently, it was reported (Zagrean-Tuza et al., 2020) quercetin had a higher antioxidant activity with respect to 3-O-glycosides (i.e. isoquercitrin). Previous toxicological studies of isoquercitrin containing formulations (alpha-glycosyl isoquercitrin) reported its safety, non-carcinogenicity, and non-genotoxicity (Nyska et al., 2016; Salim et al., 2004; Hasumura et al., 2004), and it has also tested negative in in-vivo mammalian micronucleus and chromosomal aberration assays, combined micronucleus and comet assays (Hobbs et al., 2018).

In the present work, the toxicological studies are performed on a novel isoquercitrin containing formulation to demonstrate its safety. The schematic illustration of proprietary isoquercitrin-γ-cyclodextrin inclusion complexes (IQC-γCD) [SunActive® QCD/EN; isoquercitrin content: 20% (w/w); solubility: nearly 1000 times of pure isoquercitrin; manufactured by Taiyo Kagaku Co., Ltd., Mie, Japan], wherein quercetin glycosides are included in γ-cyclodextrin through controlled enzymatic hydrolysis of rutin as reported elsewhere (Kapoor et al., 2021a; Moriwaki et al., 2020). Because of the inclusion of the isoquercitrin inside the γ-CD cavity, the IQC-γCD inclusion complex is readily soluble in water (Kapoor et al., 2021a), and isoquercitrin released from IQC-γCD inclusion complex is absorbed well (Kapoor et al., 2021c).

A 13-week subchronic dietary GLP-compliant toxicity study in Sprague-Dawley (SD) rats resulted in a no observable adverse effect level (NOAEL) of 5.0% IQC-γCD inclusion complex in the diet (3338.6 mg/kg/day) for male and 3.0% IQC-γCD inclusion complex in the diet (2177.3 mg/kg/day) for female rats. No IQC-γCD inclusion complex related adverse clinical or pathological findings were observed, hence confirming the safe use of IQC-γCD inclusion complex as a functional food, food additive, and natural ingredient at these levels (Kapoor et al., 2021b). However, the mutagenic and genotoxicity of the IQC-γCD inclusion complex have not yet been scientifically evaluated.

The Ames test, and combined micronucleus/comet assays are the most widely applied test methods available to evaluate the mutagenicity/genotoxicity of the flavonoids due to their simplicity, reliability, and proven suitability for toxicology potential evaluation (Lorge et al., 2007; Fenech, 2008). Therefore, the present study was undertaken to investigate mutagenicity and in-vivo genotoxicity of IQC-γCD inclusion complex by using the bacterial reverse mutation assay and a combined micronucleus and comet assay, respectively, under the guidelines of the Organisation for Economic Co-operation and Development (OECD). These studies can provide useful information on the safety of this novel functional product IQC-γCD inclusion complex.

MATERIALS AND METHODS

The genotoxicity and mutagenicity evaluation of the present study was conducted according to the Organisation for Economic Co-operation and Development (OECD) guidelines and were in line with the Ordinance on Good Laboratory Practice (GLP) for Nonclinical Safety Studies of Drugs (Ordinance No. 21 of the Ministry of Health, Labour and Welfare, Japan, March 26, 1997, amended by No. 114 of June 13, 2008 (MHLW, 1997), and “OECD Principles of Good Laboratory Practice”, November 26, 1997) (OECD, 1997a, 1997b).

Dietary test material

The test substance is a proprietary formulation of a patented composition containing a isoquercitrin-γ-cyclodextrin inclusion complex (IQC-γCD), wherein quercetin glycosides (i.e. isoquercitrin; CAS No. 21637-25-2) are included in γ-cyclodextrin through controlled enzymatic hydrolysis of rutin (extract from the bud/flowers of Sophora japonica Linné plant; listed in Japan specification and standard for food additive (http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/pages/spec.stand.fa) after the loss of terminal rhamnose (Moriwaki et al., 2020) [SunActive® QCD/EN; isoquercitrin content: 20% (w/w); solubility: nearly 1000 times of pure isoquercitrin manufactured by Taiyo Kagaku Co., Ltd., Japan]. The chemical structure of the IQC-γCD inclusion complex is displayed in Fig. 1.

Fig. 1

The schematic structural representation of isoquercitrin-γ-cyclodextrin (IQC-γCD) inclusion complex.

The dose formulation was analyzed for achieved concentrations under the GLP conditions. IQC-γCD inclusion complex was suspended in water for injection (Japanese Pharmacopoeia, Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) for the Ames test and a combined micronucleus and comet assay. Also, it was verified that all test substance doses were stable during the experimental periods, and the results of analyses of the dose formulations used for in-vivo studies met the acceptance criteria. Prepared doses were stored in a refrigerator (acceptable range: below 10°C), airtight in hermetic containers, and protected from light.

Chemicals and reagents

The water for injection (Otsuka Pharmaceutical Factory Inc.) was chosen for negative control (vehicle) because the homogenous suspension of the test dietary material IQC-γCD inclusion complex could be prepared. The mutagens 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), sodium azide (SAZ), 2-aminoanthracene (2AA), and benzo [α] pyrene (B [α] P) of very high purity were supplied by FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, and were used as the positive control for Ames test. Further, 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino] acridine·2HCl (ICR-191) was purchased from Polysciences, Inc. (Warrington, UK). Agar (Bacto-Agar) was purchased from Becton, Dickinson and Company (NJ, USA), and Agar (Taiyo-Agar) was obtained from SSK Sales Co., Ltd. (Shizuoka, Japan) Dimethyl sulfoxide (DMSO) and other general reagents sodium chloride, hydrochloric acid (6N), ethanol (purity: 99.5%) D-biotin, L-histidine hydrochloride monohydrate, L- tryptophan, phosphate buffer (0.1 mol/L; pH 7.4) were supplied by Wako Pure Chemical Corporation, Japan. Nutrient Broth No. 2 (Oxoid, Ltd., Altrincham, UK) was used after diluting in purified water at the concentration of 2.5 wt.% followed by sterilization in an autoclave (121°C for 20 min). Positive controls ethyl Methanesulfonate (EMS) for comet assay and Mitomycin C (MMC) for micronucleus assay were purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan, and Kyowa Kirin Co., Ltd., Tokyo, Japan, respectively. Sodium hydroxide, Agarose (A9539), Trizma base, and Triton X-100 were purchased from Merck, Tokyo, Japan. Hank’s balanced salt solution (HBSS), Dulbecco’s phosphate buffer (DPBS; Ca⁺², Mg⁺² and phenol-red free), and fetal bovine serum were supplied by Thermo Fisher Scientific, Inc. (MA, USA). Ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) was obtained from Dojindo Molecular Technologies, Inc., Tokyo, Japan, and used as 0.2 M EDTA solution (pH 10; adjusted by 10 M sodium hydroxide solution). Mincing buffer was prepared from HBSS, 0.2 M EDTA-2Na solution and DMSO mixed in an 8:1:1 volume ratio, respectively. A 1.5% agarose solution and 0.5% low melting agarose solution were obtained using DPBS with mild heating. Lysis buffer with different concentrations (2.5 M, 100 mM, and 10 mM) were prepared using sodium chloride, EDTA-2Na, and Trizma base in the water of injection (pH 10; adjusted by 10 M sodium hydroxide solution), and the solution was stored in a refrigerator. Prior to use, DMSO (10% v/v) and Triton X-100 (1% v/v) were added, respectively. Electrophoresis buffer (pH > 13) was obtained by mixing 10 M sodium hydroxide solution and 0.2 M EDTA-2Na solution in water for injection, and final concentrations of sodium hydroxide and EDTA-2Na were adjusted at 300 mM and 1 mM, respectively. Neutralization buffer (0.4 M) was prepared by dissolving Trizma base in water for injection (pH 7.5; adjusted by 6N hydrochloric acid) and stored in a refrigerator.

Bacterial reverse mutation assay (Ames test)

The mutagenicity assay of IQC-γCD inclusion complex was conducted according to published methods (Ames et al., 1975; Maron and Ames, 1983; Gatehouse et al., 1994; Mortelmans and Zeiger, 2000) following the OECD test guidelines 471 of the bacterial reverse mutation test (OECD, 1997c; OECD, 2020). The Ames Salmonella/microsome mutagenicity assay is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations.

Confirmation of tester strains

Tester strains (bacteria) Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2uvrA were originally obtained from the Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Health Sciences, Tokyo, Japan. All strains were confirmed for maintenance of genetic markers before use in this study. S. typhimurium TA98 (allele: His D3052; DNA target: -C-C-C-C-C-C-) and TA1537 (or TA97, allele: His D6610; DNA target: -C-G-C-G-C-G-C-G-) were used as the frameshifts type reversion event. While, both S. typhimurium TA100 and TA1535 (allele: His G46, DNA target: -G-G-G-) were used as the base-pair substitution type, and Escherichia coli WP2uvrA (or TA102, Allele: His G428; DNA target: TAA) was used as the base-pair transitions/ transversions type events (an ochre mutation). Details of tester strains used could be followed elsewhere (Levin et al., 1982a, 1982b; Isono and Yourno, 1974; Barnes et al., 1982). The reason for the selection of the aforementioned tester strains is that strains are susceptible to mutagens and used most generally in mutagenicity studies using microorganisms. The tester strains were cultured and DMSO added (0.7 mL per 8.0 mL of strain). The solution was divided into aliquots (0.3 mL each), rapidly frozen by dry ice-acetone, and then preserved in an ultra-deep freezer at –70°C. At the time of use, the aliquots were thawed at ambient conditions. The frozen bacterial strains were subjected to a characteristics test including amino acid requirement, membrane-variation rfa- characteristics, drug resistance factor R-factor plasmid, sensitivity to UV light, rate of bacterial multiplication, negative control values, and positive control values (data not shown), and used after confirming that their specific characteristics were retained.

Metabolic activation

S9 microsomal fraction derived from male Sprague Dawley rat strain (Tsukuba Research Institute, BOZO Research Center Inc., Ibaraki, Japan) was used to prepare S9 Mix. Metabolic activation was provided through inducers phenobarbital (PB) and 5, 6-benzoflavone (BF) by intraperitoneal administration method with added co-enzyme Cofactor FA (Tsukuba Research Institute, BOZO Research Center Inc.) in a volume ratio of 1:9. The composition of S9 mix (per mL) was distilled sterile water (0.9 mL), S9 (0.1 mL), MgCl₂ (8 μmol/mL), KCl (33 μmol/mL), glucose 6-phosphate (5 μmol/mL), reduced nicotinamide adenine-dinucleotide phosphate (NADPH, 4 μmol/mL), reduced nicotinamide adenine-dinucleotide (NADH, 4 μmol/mL), and sodium phosphate buffer (pH 7.4, 100 μmol/mL). The mutagenicity was determined by the incorporation method in the presence and absence of S9 metabolic activation (Khoury et al., 2016). The background data of the S. typhimurium and E. coli tester strains based on the pre-incubation method for the bacterial reverse mutation test with or without S9 mix metabolic activation is presented in Table S1 (see supplementary information).

Experimental procedures

The test formulations for the dose-finding test as well as the first and second main Ames tests were prepared separately. For the dose-finding test, the IQC-γCD inclusion complex (270.0 mg) was mixed with 5.4 mL of water for injection (i.e. 50 mg/ mL; isoquercitrin content: 10.3 mg/mL), and was sterilized by passing through a membrane filter (MiLLEX GV Filter Unit 0.22 μm). The solution was diluted 4 times using a common ratio of 4 to prepare a total of 5 dose concentrations viz. 50, 12.5, 3.13, 0.781 and 0.195 mg/mL (10.3, 2.56, 0.641, 0.160 and 0.040 mg/mL as isoquercitrin content). Similarly, the test formulations for the first and second main Ames tests were prepared by diluting their respective mother solution by 4 times using a common ratio of 2 to prepare a total of 5 dose concentrations viz. 50, 25, 12.5, 6.25 and 3.13 mg/mL (10.3, 5.13, 2.56, 1.28 and 0.641 mg/mL as the isoquercitrin content). All test dose formulations were prepared at the time of use under the UV absorption film-coated fluorescent lamp, and there was no reaction resulting in heat or gas generation.

An aliquot of the test formulation dose (0.1 mL), vehicle (negative control), or positive control solution was separately put into a sterilized test tube, 0.5 mL of S9 mix or 0.5 mL of phosphate buffer (0.1 mol/L) in the absence of S9 mix, and 0.1 mL of bacterial culture suspension (after pre-culturing the preserved test bacterial strains in Nutrient Broth No. 2 culture medium; S. typhimurium TA strains: 20 μL in 10 mL; E. coli WP2uvrA strain: 10 μL in 10 mL) were added. The converted viable cell count for each bacterial strain is given in Table 1 for the dose-finding test along with the first (Experiment 1) and second (Experiment 2) main mutagenicity tests. The concentrations of positive control substances either with or without S9 metabolic activation used in this study are listed in Tables 2 and 3. The content of each sterilized test tube was mixed and incubated for 20 min at 37°C while continuous shaking. After incubation, a volume of 2 mL of molten top agar was added to each test tube (a soft liquid containing 0.6 wt.% Agar and 0.6 wt.% NaCl was sterilized in an autoclave at 121°C for 20 min, and then the 0.5 mmol/L D-biotin-L-histidine-L-tryptophan solution added in the ratio of 1:10 to the soft agar solution to prepare top agar), mixed and overlaid uniformly onto the surface of a minimal glucose agar plate (Vital Media AMT-S Medium; Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan), and allowed to solidify. The plates were placed in an incubator inversely, and cultured and protected from light for 48-hr at 37°C before scoring the number of revertant colonies.

Table 1. Converted viable cell count for each bacterial tester strain used in reverse mutation assay (Ames test).

Bacterial tester strains S. typhimurium	Viable cell count (x 109 cells/ mL)
	Dose Finding	EXP 1	EXP 2
TA100	2.62	2.47	2.35
TA1535	3.91	3.94	2.35
WP2uvrA	6.95	6.85	6.71
TA98	4.23	4.23	4.22
TA1537	6.98	6.51	8.12

Table 2. Bacterial reverse mutation assay (Ames test) results of IQC-γCD inclusion complex using base-pair substitution type S. typhimurium tester strains with and without S9 metabolic activation in both first and second mutagenicity tests.

With (+) or Without (−) S9 Mix	Test article dose (μg/plate)	Isoquercitrin equivalence dose (μg/plate)	Number of revertants (Number of colonies / plate)
			TA100										TA1535										WP2uvrA
			EXP 1			EXP 2			Average				EXP 1			EXP 2			Average				EXP 1			EXP 2			Average
9 Mix (−)	Negative control (Water)		117	±	5.5	133	±	6.1	125	±	10.0		10	±	0.6	10	±	1.0	10	±	0.8		23	±	2.1	29	±	1.2	26	±	3.6
	313	64.1	130	±	2.3	161	±	7.6	145.5	±	17.7		9	±	2.9	11	±	3.8	10	±	3.3		25	±	5.0	32	±	4.6	28.5	±	5.8
	625	128	161	±	22.0	217	±	14.7	189	±	34.8		9	±	2.6	9	±	3.2	9	±	2.6		31	±	9.5	33	±	3.2	32	±	6.4
	1250	256	197	±	5.9	223	±	13.5	210	±	17.2		9	±	0.6	10	±	1.0	9.5	±	0.8		21	±	2.9	30	±	5.6	25.5	±	6.5
	2500	513	223	±	16.7	253	±	7.0	238	±	19.9		8	±	3.6	14	±	2.0	11	±	4.2		28	±	2.9	32	±	6.1	30	±	4.9
	5000#	1025	297	±	9.9	433	±	38.8	365	±	78.67		10	±	3.1	14	±	3.0	12	±	3.6		36	±	5.0	35	±	6.9	35.5	±	5.4
S9 Mix (+)	Negative control (Water)		125	±	5.0	137	±	18.5	131	±	13.7		9	±	1.5	8	±	1.2	8.5	±	1.3		25	±	3.8	38	±	2.5	31.5	±	7.7
	313	64.1	140	±	3.8	189	±	11.5	164.5	±	28.3		10	±	1.5	14	±	3.1	12	±	3.1		31	±	0.6	35	±	5.3	35	±	3.9
	625	128	218	±	3.0	264	±	6.7	241	±	25.8		11	±	4.7	12	±	1.7	11.5	±	3.3		33	±	7.0	35	±	2.3	33	±	4.8
	1250	256	296	±	6.8	350	±	34.5	323	±	36.7		14	±	1.5	14	±	1.7	14	±	1.5		28	±	4.5	30	±	1.5	29	±	3.1
	2500	513	277	±	22.1	386	±	24.0	331.5	±	63.2		13	±	2.0	13	±	4.6	13	±	3.2		39	±	7.9	35	±	4.5	37	±	6.1
	5000#	1025	513	±	10.8	636	±	16.2	574.5	±	68.3		22	±	2.6	17	±	2.5	19.5	±	3.5		29	±	8.6	33	±	5.7	31	±	7.0
Positive control S9 Mix (−)	Name		AF-2										SAZ										AF-2
	Dose (μg/plate)		0.1										0.5										0.01
	Number of colonies/plate		608	±	7.5	592	±	48.8	600.4	±	32.4		294	±	48.8	213	±	11.3	253.5	±	54.5		101	±	17.0	82	±	5.0	91.5	±	15.2
Positive control S9 Mix (+)	Name		B[a]P										2AA										2AA
	Dose (μg/plate)		5.0										2.0										10.0
	Number of colonies/plate		1266	±	87.6	1282	±	20.3	1274	±	57.5		222	±	6.2	298	±	36.1	260	±	47.5		585	±	52.5	603	±	20.1	594	±	36.8

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide.

SAZ: Sodium azide.

B[a]P: Benzo[a]pyrene.

2AA: 2-Aminoanthracene.

^#: Precipitation was observed on the surface of agar plates.

Mean and standard deviation of counted colony numbers are of three plates.

Average of both experiment 1 & experiment 2 of mutagenicity tests.

Table 3. Bacterial reverse mutation assay (Ames test) results of IQC-γCD inclusion complex using frameshift type S. typhimurium tester strains with and without S9 metabolic activation in both first and second mutagenicity tests.

With (+) or Without (−) S9 Mix	Test article dose (μg/plate)	Isoquercitrin equivalence dose (μg/plate)	Number of revertants (Number of colonies / plate)
			TA98										TA1537
			EXP 1			EXP 2			Average				EXP 1			EXP 2			Average
S9 Mix (−)	Negative control (Water)		22	±	4.4	18	±	3.2	20	±	4.0		9	±	1.0	7	±	1.2	8	±	1.3
	313	64.1	26	±	4.5	23	±	1.5	24.5	±	3.3		10	±	2.0	9	±	4.2	9.5	±	2.9
	625	128	30	±	11.2	33	±	1.5	31.5	±	7.5		15	±	0.6	13	±	2.1	14	±	1.8
	1250	256	71	±	8.3	86	±	8.3	78.5	±	10.8		18	±	2.5	28	±	1.5	23	±	5.4
	2500	513	119	±	13.5	136	±	6.7	127.5	±	13.2		42	±	1.5	31	±	5.7	36.5	±	7.1
	5000#	1025	190	±	9.0	221	±	1.7	205.5	±	17.9		48	±	7.9	42	±	2.5	45	±	6.3
S9 Mix (+)	Negative control (Water)		27	±	5.6	27	±	6.1	27	±	5.2		8	±	0.6	9	±	2.1	8.5	±	1.4
	313	64.1	70	±	10.0	63	±	1.0	66.5	±	7.5		9	±	1.2	13	±	2.5	10.5	±	2.8
	625	128	128	±	11.6	112	±	12.7	120	±	13.7		14	±	1.5	17	±	0.6	15.5	±	1.9
	1250	256	159	±	17.7	223	±	7.8	191	±	36.8		23	±	2.1	30	±	4.0	26.5	±	4.5
	2500	513	238	±	9.2	348	±	11.0	293	±	60.9		47	±	5.7	52	±	10.4	49.5	±	7.9
	5000#	1025	313	±	40.4	373	±	30.7	343	±	45.7		59	±	1.0	61	±	7.4	60	±	4.9
Positive control S9 Mix (−)	Name		AF-2										ICR-191
	Dose (μg/plate)		0.1										0.5
	Number of colonies/plate		309	±	9.0	362	±	40.3	335.5	±	38.9		1256	±	37.1	1649	±	24.8	1452.5	±	217.5
Positive control S9 Mix (+)	Name		B[a]P										B[a]P
	Dose (μg/plate)		5.0										2.0
	Number of colonies/plate		296	±	25.5	320	±	16.3	308	±	23.3		91	±	5.5	90	±	9.0	90.5	±	6.7

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamid.

ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine·2HCl.

B[a]P: Benzo[a]pyrene.

2AA: 2-Aminoanthracene.

^#: Precipitation was observed on the surface of agar plates.

Mean and standard deviation of counted colony numbers are of three plates.

Average of both experiment 1 & experiment 2 of mutagenicity tests.

In the dose range-finding experiment, all strains were treated with 5 concentrations of IQC-γCD inclusion complex in the presence and absence of S9 mix (5000 μg/plate top concentration) alongside suitable positive control substances and negative control (vehicle). Since no overt toxicity occurred in the dose range-finding assay (see Table S2 in supplementary information), isoquercitrin containing IQC-γCD inclusion complex was tested at a similar top concentration of 5000 μg/plate with a modified range of concentrations in experiment 1, and experiment 2 with or without S9 mix in all strains studied alongside positive and negative controls as described above (also see Tables 2 and 3 for concentrations). The Ames test assay was duplicated as per the OECD 471 test guideline (OECD, 1997c, 2020). After the 48-hr incubation period, revertant colonies per plate were counted using an automatic colony counter (Colony Analyzer CA-11D, System Science Co., Ltd., Tokyo, Japan). Toxicity and solubility were assessed visually and macroscopically by the presence/absence of precipitated test substance. Further, the presence/absence of growth inhibition of the bacteria was checked using a stereomicroscope. Test validation criteria for the positive control response was described as where the mean number of revertants per plate was greater than 2-fold that of the negative control means the number of revertants as well as reproducible in a follow-up experiment without any bacterial contamination in the sterility test.

In-vivo micronucleus and comet assay

The toxicity study guidelines were in accordance with “Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use”, Notification 0920, Article No. 2 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Japan, September 20, 2012 (PARJ, 2015). In-vivo micronucleus and comet assay were conducted according to recommended OECD test guidelines. The “OECD Guideline for the Testing of Chemicals 489: In-vivo Mammalian Alkaline Comet Assay”, revised July 29, 2016 (OECD, 1997d, 2016b), and “OECD Guideline for the Testing of Chemicals 474: Mammalian Erythrocyte Micronucleus Test”, revised July 29, 2016 (OECD, 1997a, 1997b, 2016a), were followed. The combined micronucleus/comet studies were performed according to “The Ordinance on Good Laboratory Practice for Nonclinical Safety Studies of Drugs”, Ordinance No. 21 of the Ministry of Health and Welfare, Japan, March 26, 1997 (MHW, 1997), and “OECD Principles of Good Laboratory Practice”, OECD: November 26, 1997 (OECD, 1997a, 1997b).

Animals husbandry and nourishing

The in-vivo experiments were performed under the authorized Guidelines for Proper Conduct of Animal Experiments, Science Council of Japan (June 1, 2006). The combined micronucleus /comet studies were conducted under the approval (No. G190172) of the Institutional Animal Care and Use Committee (IACUC). Moreover, all animals received humane care for the protection of animals utilized for scientific purposes in concordance with the Animal Welfare Act Regulations, 9 CFR 1-4, and the Guide for the Care and Use of Laboratory Animals (ILAR, 2011). Also, the studies were conducted in compliance with the “Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain” (Notification No. 88 of the Ministry of the Environment, Japan, April 28, 2006; revised as Notification No. 84, August 30, 2013) (MOE, 2013), and “Act on Welfare and Management of Animals” (Act No. 105 of October 1, 1973; revised as Act No. 38 of June 12, 2013) (WMA, 2013).

Thirty-three male Sprague-Dawley (SD) rats [strain Crl: CD (SD)], aged 7 weeks, were purchased from Charles River Laboratories Japan, Inc., Kanagawa, Japan. The animals, in groups of two or three, were housed in solid-floored plastic cages (W 440 × D 275 × H 180 mm) with bedding (ALPHA-dri, Shepherd Specialty Papers, Inc., MI, USA) to allow recording the clinical observations, including any abnormality in the external appearance, nutrition condition, posture, behavior and excretions, and to avoid bias from hierarchical stress. The housing facility was controlled [temperature 23°C ± 3°C; relative humidity at 50% ± 20%; air ventilation at 10 to 15 times per hour, and 12-hr illumination (7:00 a.m. to 7:00 p.m.)]. The animals were allowed free access to pelleted diet CR-LPF (γ-irradiated, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water. Appropriate environmental enrichment was given in accordance with the guideline of the IACUC. Moreover, rats were allowed a 1-week quarantine and acclimation period before the start of the study. Animals were 8 weeks of age with body weight between 298 to 344 g (mean 318 g; ± 20% within the mean value) at the start of the study. Healthy rats without abnormalities were assigned to each group by individual body weights to ensure uniformity of group body weight means by block randomization.

Study design and sample collection

Six groups of male SD rats (5 rats per group) were administrated IQC-γCD inclusion complex at the dose level of 500, 1000, and 2000 mg/kg bw per day in water, a negative control vehicle (physiological saline water) or the positive control compound for comet assay, ethyl methanesulfonate (20 mg/mL) solution (336 μL of EMS i.e. 406.6 mg in 20 mL of physiological saline water) or the positive control compound for micronucleus assay, mitomycin C (0.2 mg/mL) solution (10 mg MMC in 25 mL of water of injection, and further diluted in 20 mL of physiological saline in 20/20 v/v ratio to make 0.2 mg/mL MMC solution).

IQC-γCD doses were confirmed by HPLC (Waters Corporation, MA, USA) and checked for their stability. They were administered by gavage (oral) using a flexible stomach tube at a dose volume of 10 mL/kg for 3 consecutive days (approximately 24–26 hr intervals). The negative control animals received the vehicle (physiological saline water) alone in the same route. The comet assay positive control animals received EMS at 200 mg/kg bw/day by oral gavage at a dose volume of 10 mL/kg for 2 consecutive days, on day 2 and day 3 of the experiment. Whereas, the micronucleus positive control animals received a single intraperitoneal dose of MMC solution at 2 mg/kg/day using a 26-G injection needle at the dose volume of 10 mL/kg on day 2 of the experiment.

In-life observations for animals in each group, including clinical signs, abnormalities in appearance, nutritional condition, posture, behavior, and excretions, etc., were observed two or three times daily, and the body weights were recorded every day.

Comet assay: Sample collection and procedure

Two to six hours after the final dose on day 3 of the experiment, the animals were euthanized by exsanguination via the abdominal aorta under anesthesia by isoflurane inhalation. The liver was dissected from animals of the test article intake groups, negative control group, and positive control EMS group.

Small pieces of the liver tissue from the left lateral lobe were weighed (0.05–0.10 g) and washed extensively with the ice-cold mincing buffer to remove the blood. After that, liver tissue was homogenized using a homogenizer and passed through a cell strainer (pore size: 40 μm) to obtain a single-cell suspension for performing the comet assay (Corcuera et al., 2015; Mellado-García et al., 2016). The single-cell suspension was mixed with 0.5% low melting agarose gel (Lonza, Tokyo, Japan). Then, the mixture was dropped on two places on the slide (two spots per slide, 3 slides per animal; 6 spots per animal in total) coated with 1.5% agarose gel and solidified on ice. These slides were exposed to ice-cold lysis buffer (immersed), protected from light, and placed in a refrigerator overnight. The slides were placed in a submarine type electrophoresis tank (Bio Craft, BE-540) filled with electrophoresis buffer, and incubated for at least 20 min (unwinding). Then, electrophoresis was performed for 20 min with an electric potential from 0.7 to 1.0 V/cm. After that, the slides were immersed in neutralization buffer to neutralized DNA followed by immersion in ethanol (fixing) and air-dried. The slides were stored at room temperature and low humidity in a desiccator until staining. Just before scoring, the slides were stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific KK, Tokyo, Japan), and scored using an image analysis system (Comet assay IV, Instem, UK) using a fluorescence microscope equipped with a CCD camera. Images of at least one hundred and fifty randomly selected nuclei per animal (50 nuclei per spot, therefore a total of 3 spots) were examined. The magnitude of DNA migration was characterized using the % tail DNA endpoint analysis, wherein the intensity of all tail pixels divided by the total intensity of all pixels in the comet was expressed as a percentage. Following the test guideline recommendation, the median value for the % tail DNA (= Tail intensity) was calculated for each spot, and the mean value of the median of scored comets of 3 spots was treated as an individual value for each animal and used to calculate the dose group means values. The % DNA in the tail represents DNA strand breaks and oxidized damage in DNA bases.

Erythrocyte micronucleus assay: Sample collection and procedure

Samples of micronucleus assay were collected from each animal and immediately processed. After dissecting the liver for the comet assay, the right femur was dissected from the negative control group and test article intake groups. The right femur was also dissected from the micronucleus positive control MMC group after the euthanasia by exsanguination via the abdominal aorta under anesthesia by isoflurane inhalation.

After dissection, both the distal and proximal ends of the femur were cut off and the bone marrow cells were flushed, suspended in fetal bovine serum (FBS, 0.2 to 0.4 mL), centrifuged (1000 rpm), and re-suspended in FBS by pipetting. The in duplicate cell suspension were transferred onto a slide, smeared, air-dried, fixed with methanol, and air-dried again and stored at room temperature.

For each animal, the better smear was stained with Acridine Orange just before performing the bone marrow extensions. After straining, the slides were observed under a fluorescent microscope at the magnification of 600-fold. A total of 500 erythrocytes per sample were scored to determine the number of polychromatic erythrocytes (PCE; immature erythrocytes) and normochromatic erythrocytes (NCE). The number of incidences of PCE (PCE% incidence) among a total of 500 erythrocytes (PCE + NCE) and PCE/NCE relative ratio was reported. PCE was visible as bright orange enucleated erythrocytes, while NCE was visible as smaller dark green enucleated erythrocytes. Further, a total of 4000 PCE per animal were counted and the number (incidence) of micronucleated polychromatic erythrocytes (MNPCE) per 4000 PCE was determined, wherein the incidence of MNPCE per sample was expressed as the % of MN/PCE.

Statistical analysis

All statistical analyses were performed using an integrated package Statistical Analysis System, Release 9.1.3 (SAS Institute Inc., Cary, NC, USA). For the comet assay mean ± SD of the medians was calculated for each group. The % tail DNAs of the negative control group and the comet positive control EMS group were compared using a two-sample Student’s t-test (one-tailed). For the % tail DNAs of the negative control group and test article intake groups, a simple linear regression analysis was applied to assess the trend involved with the possible dose-response relationships. Dunnett’s test (one-tailed) was performed for pairwise comparisons of each test article intake group with the negative control group. All tests were performed with one-side risk for the increased response with increasing dose. Probability values of p ≤ 0.05 were considered significant.

The result of the micronucleus assay is presented as the mean ± SD for each group. For the MNPCE incidence, a pairwise comparison was performed between the negative control group and the micronucleus positive control MMC group, and between each test article intake group and the negative control group using Fisher’s exact test (one-tailed). All tests were interpreted with a one-sided risk for increased incidence. In case of a significant increase noted in any test article intake group, the dose-dependency assessment was recommended by the Cochran-Armitage trend test (if required). For the proportion of PCEs, data from the negative control group and test article intake groups were assessed by the non-parametric Bartlett’s test for homogeneity of variance (level of significance: p ≤ 0.01). Then Dunnett’s test (two-tailed) was performed for pairwise comparisons of each test article intake group with the negative control group. Probability values of p ≤ 0.05 and p ≤ 0.01 were considered significant.

Criteria for a positive outcome in the in-vivo comet assays and in-vivo micronucleus assays were (i) at least one statistically significant (p ≤ 0.05) dose group compared to the negative control group, (ii) a dose group falling outside the range of laboratory historical data (for micronucleus assay only), and (iii) a statistically significant trend test (p ≤ 0.05; dose-dependency). A test was considered equivocal if only one or two of the above conditions were satisfied (OECD, 2016a, 2016b).

RESULTS

Bacterial reverse mutation test (Ames test) results

The mutagenicity assay was conducted to evaluate the potential of the IQC-γCD inclusion complex to induce gene mutation in bacteria. Assay acceptability was confirmed by positive and negative controls falling within normal ranges and adequate numbers of IQC-γCD inclusion complex treated plates were scored at each dose level, in all bacterial strains. A dose range finding assay using five S. typhimurium tester strains with or without metabolic activation with S9 mix is shown in Table S2 (supplementary information). These preliminary results provided evidence of precipitation at 5000 μg/plate in all strains; they showed a dose-correlated increase in the number of mutant colonies more than twice that of the negative control group. Therefore, a top concentration of 5000 μg/plate in all strains with or without S9 mix metabolic activation, was chosen for this study according to recommendations in the guidelines of OECD (OECD, 1997b, 2020). The initial mutagenicity test used dose levels of IQC-γCD inclusion complex ranging from 19.5 to 5000 μg/plate in the dose-finding assay. Following these dose levels, a top concentration of 5000 μg/plate for all tester strains was used, either in the absence or presence of S9 mix metabolic activation. Furthermore, in the first and second mutagenicity tests, a revised dose concentration interval was employed covering the range 313–5000 μg/plate to better examine dose levels of IQC-γCD inclusion complex approaching the maximum test dose level. The effects of IQC-γCD inclusion complex on base-pair substitution type (TA100, TA1535, and WP2uvrA) and frameshift type (TA98 and TA1537) tester strains with or without metabolic activation with S9 mix are shown in Tables 2 and 3, respectively.

Both first (EXP 1) and second (EXP 2) mutagenicity test experiments produced results similar to those of the dose-finding test. Additionally, the mean revertants/plate of both the first and second mutagenicity test experiments of bacterial reverse mutation assays of IQC-γCD inclusion complex either in the absence or presence of S9 mix metabolic activation using representative tester strains of bacteria presented in Fig. 2 further confirms the absence of cytotoxicity or mutagenicity.

Fig. 2

Bacterial reverse mutation assay of IQC-γCD inclusion complex in the absence of (-S9) and presence (+S9) of metabolic activation using base-pair substitution and frameshift types S. typhimurium tester strains of bacteria. (Average values of two mutagenicity tests are presented; results of positive control are shown in Tables 2 and 3).

Compared with the negative control, the positive controls with (B[α]P and 2AA) or without (AF-2, SAZ, and ICR-191) S9 mix for the tester strains produced the increased revertant effect. In the Ames test, similar to the results obtained in preliminary dose-finding studies (Table S2), a remarkable increase in the number of revertants was observed in all positive control groups compared to the negative control in both first and second mutagenicity test experiments, demonstrating the reproducibility of the positive response. Although the positive controls induced a positive response in all tester strains, the mean of the scored revertant colonies per plate was within the historical reference ranges of the laboratory. The negative control (vehicle) and positive controls for all experiments met acceptance criteria.

Exposure to varying dose levels of IQC-γCD inclusion complex induced a 2-fold or greater increase in the number of revertant colonies per plate relative to the negative (vehicle) control group in the base-pair substitution type TA100 strain (dose level 5000 μg/plate only), and the frameshift type TA98, and TA1537 tester strains (dose levels of 1250 μg/plate and above) without S9 mix metabolic activation. Whereas, with S9 mix metabolic activation the increased score (2-fold and higher) of revertant colonies per plate relative to the negative control was observed for TA100 strain (dose level of 1250 μg/plate and above) and TA1535 strain (dose level 5000 μg/plate only). On the other hand, frameshift type TA98, and TA1537 tester strains showed the above 2-fold increase in revertant colonies per plate with S9 mix metabolic activation, at all dosage levels studied and at dose levels of 1250 μg/plate and above, respectively. In the WP2uvrA strain, there was no increase in revertant colonies greater than 2-fold over negative control at any dose levels tested either in the absence or presence of S9 mix metabolic activation. Further, the specific activity values (number of revertant colonies more than 2-fold of the negative control) are presented in Table S3 (see supplementary information). Specific activity is calculated as the number of revertant colonies per 1 mg of test article IQC-γCD inclusion complex using the formula:

Specific activity (Rve/mg) = (No. of colonies of dose concerned − No. of colonies of negative control) x 1000 / Dose concerned (μg/plate)

The maximum specific activity (170 Rve/mg) was estimated for S. typhimurium TA100 strain at a dose level of 1250 μg/plate with S9 mix metabolic activation in the second mutagenicity test experiment. The above maximum specific activity of dose level concerned was associated with a 2.55 fold increase in revertant colonies over negative control (vehicle), however, the increase was considered to be biological irrelevant. Similar results were obtained in the preliminary dose-finding assay, demonstrating the reproducibility of the responses. All dose levels of IQC-γCD inclusion complex for studied tester strains were without any considerable revertants change, and the increase in revertants/plate indicated a relationship with varying dosage levels and were considered dose-dependent. Further, a dose-dependent response along with a corresponding doubling of revertants observed was considered equivocal, and the effect was not considered biologically relevant. Thus, the results indicate that no obvious mutagenic activity of IQC-γCD inclusion complex, up to a dose level of 5000 μg/plate, and no evidence of toxicity (cytotoxic or mutagenic) could be declared.

Comet assay results

Clinical signs of toxicity were completely absent during the study in any of the dose groups. No abnormal behavior, death, or evidence/symptoms of pain, as well as no significant body weight change, were observed even at the maximum dose at 2000 mg/kg bw. Slight dose-related differences in body weight gain were observed at all doses of IQC-γCD inclusion complex, and values were always comparable to that of the negative control (vehicle) group, except for the EMS positive control (Table 4).

Table 4. Summary of body weight changes in SD rats administrated IQC-γCD inclusion complex in diet.

Dose (mg/kg bw)	Body weight change
	(g)			(%)
Negative control	19.2	±	3.27	6.01	±	0.88
500	11.6	±	6.15	3.62	±	1.83
1000	18.2	±	3.77	5.74	±	1.24
2000	20.6	±	4.39	6.45	±	1.20
Positive control MMC#	15.2	±	5.89	4.77	±	1.86
Positive Control EMSδ	–5.0	±	10.54	–1.49	±	3.31

N = 5 animals in each group; Mean ± S.D. bw = body weight.

^#2 mg/kg bw, Dose level of MMC.

^δ200 mg/kg bw, Dose level of EMS.

No significant difference in any test groups from the negative control group.

The results of the comet assay for the assessment of DNA damage in liver tissue of SD rats administrated with IQC-γCD inclusion complex are presented in Fig. 3. The results indicate there was no significant increase in the induction of DNA strand breaks in the liver at any dose compared to the negative control (vehicle) group. The group mean % tail DNA values for the liver of rats tested at the doses of 0, 500, 1000, and 2000 mg/kg bw/day were 2.2 ± 0.3%, 2.1 ± 0.2%, 2.2 ± 0.3% and 2.1 ± 0.3%, respectively, thus were not significantly different from the negative control group.

Fig. 3

Dose response of the exposure to IQC-γCD inclusion complex in male Sprague Dawley rats in conventional in-vivo liver comet assay. The levels are expressed as % DNA in tail. All values are expressed as mean ± SD. *(p < 0.05): significantly different from the negative (vehicle) control.

All % tail DNA values fell within the range of the concurrent negative control (vehicle) data. No statistically significant linear trend was observed. An increase in DNA damage was detected in the liver of SD rats administrated ethyl methanesulfonate (EMS; 200 mg/kg bw) as a positive control. The EMS induced a statistically significant increase (p < 0.05) in DNA damage in the liver with a value 51.0 ± 5.2%. The comet assay was considered statistically negative as the IQC-γCD inclusion complex exposure led to no induction in % tail DNA values at all doses compared to the negative control (vehicle) group.

Mammalian erythrocyte micronucleus assay results

The results of the in-vivo micronucleus (MN) assay of the IQC-γCD inclusion complex at different doses in SD rats’ bone marrow are summarized in Table 5. The SD rats treated with IQC-γCD inclusion complex at all doses exhibited a group means percentage incidence of polychromatic erythrocytes (PCE % incidence) that were similar to the negative control (vehicle) group, indicating an absence of bone marrow toxicity. No statistically significant differences (p > 0.05; Fischer exact test) were noted in PCE % incidence for any of the groups treated with IQC-γCD inclusion complex compared to the negative control group. The positive control Mitomycin C (MMC, 2.0 mg/kg bw) induced a similar decrease, and micronucleus incidences were considered consistent with the concurrent negative control (vehicle) data.

Table 5. Micronucleus assay results in SD rats administrated IQC-γCD inclusion complex in the diets. Bone marrow cytotoxicity expressed as polychromatic erythrocytes (PCE) among total erythrocytes, and the micronuclei induction expressed as MNPCE% incidence.

Doseδ (mg/kg bw)	No. of MNPCE in 4000 PCE				MNPCE% incidenceφ				No. of PCE in 500 erythrocytes				PCE% incidenceψ				PCE/NCE ratio
	Mean	±	S.D.	Min. / Max.	Mean	±	S.D.	Min. / Max.	Mean	±	S.D.	Min. / Max.	Mean	±	S.D.	Min. / Max.	Mean	±	S.D.	Min. / Max.
Negative control	5.4	±	2.3	3 / 9	0.14	±	0.06	0.08 / 0.23	320.4	±	24.9	292 / 347	64.1	±	5.0	58.4 /69.4	1.83	±	0.39	1.40 / 2.27
500	4.6	±	1.5	3 / 7	0.12	±	0.04	0.08 / 0.18	277.2	±	21.5	251 / 303	55.4	±	4.3	50.2 / 60.6	1.26	±	0.22	1.00 / 1.53
1000	6	±	2.5	4 / 10	0.15	±	0.06	0.10 / 0.25	282.2	±	43.0	236 / 328	56.4	±	8.6	47.2 / 65.6	1.37	±	0.46	0.89 / 1.70
2000	5.8	±	1.8	3 / 7	0.15	±	0.04	0.08 / 0.18	288.8	±	18.2	265 /312	57.8	±	3.6	53.0 / 62.4	1.38	±	0.21	1.13 / 1.66
Positive control MMC#	143.8	±	30.2*	104 / 173	3.60	±	0.76	2.60 / 4.33	264.4	±	52.4	215 /328	52.9	±	10.5	43.0 / 65.6	1.22	±	0.53	0.75 / 1.91

N = 5 animals in each group; bw = body weight.

^φ: Proportion (%) of micronucleated polychromatic erythrocytes (MNPCE) per 4000 polychromatic erythrocytes (PCE).

^ψ: Proportion (%) of polychromatic erythrocytes (PCE, including MNPCE) per 500 erythrocytes.

^#: 2 mg/kg bw, Dose level of MMC.

*: Significantly different from the negative control group (Fisherʼs exact test, P < 0.05, upper tailed).

^δ: No significant difference in any dose groups from the negative control group.

The percentage incidence of micronucleated polychromatic erythrocytes (MNPCE Incidence %) observed in SD rats exposed to all doses of IQC-γCD inclusion complex, were also similar to the negative control group, except the values observed for MMC positive control group (3.60 ± 0.76%) that elicited a significant increase (p < 0.05) response in the percentage of MNPCE in immature erythrocytes compared to the negative control group. Regarding the PCE/NCE ratio in SD rats, a non-significant decrease was noticed with respect to the negative control. The data demonstrated that there was no dose-dependent differences or dose-related pattern in all IQC-γCD inclusion complex treated groups. Thus, under the condition of the study, the IQC-γCD inclusion complex did not induce an increase in MNPCE in the bone marrow of SD rats following oral gavage administration of doses up to 2000 mg/kg bw/day.

DISCUSSION

The demand for healthier functional food products is on the rise, including flavonoids, particularly quercetin glycosides derived from fruits and botanicals. Therefore, systematic toxicological analyses are needed to predict the toxicity of flavonoids compounds and develop a criterion for establishing a safe dose in humans. The present work followed the regulatory guidelines to generate and evaluate the genotoxic potential of IQC-γCD inclusion complex, including through a core battery of in-vitro test of a bacterial reverse mutation assay (Ames test), and in-vivo combined assay of micronucleus and comet tests (OECD 2016a, 2016b). The Ames assay is an effective method for inducing mutations by test ingredients. In the current study, five tester bacterial strains of S. typhimurium (TA100, TA1535, WP2uvrA, TA98, and TA1537) were tested. The test plate without S9 mix as metabolic activation was to assay in-vitro genotoxicity of IQC-γCD inclusion complex self, while the plate with S9 mix as metabolic activation to evaluate in-vitro genotoxicity of IQC-γCD inclusion complex metabolites. Based on the data presented, a dose-dependent response along with a corresponding doubling of revertants observed without any significance reached was considered equivocal, and the effect was not considered biologically relevant. Thus, indicates IQC-γCD inclusion complex does not possess significant toxicity (cytotoxic or mutagenic) up to a dose level of 5000 μg/plate. Similarly, alpha-glycosyl isoquercitrin and isoquercitrin are reported to induce mutations in several bacterial strains, with or without metabolic activation, there was no evidence of the mutagenic potential of either substance detected in any of the multiple tissues examined in transgenic mice (Hobbs et al., 2018; Engen et al., 2015). However, the Ames test alone does not provide direct information on the mutagenic and carcinogenic potency of the IQC-γCD inclusion complex in mammals (humans). The mammalian in-vivo micronucleus and comet assays are especially relevant to assessing mutagenic hazards in that it allows consideration of diverse factors related to in-vivo metabolism, pharmacokinetics, and DNA repair processes (Araldi et al., 2015).

Therefore, we evaluated the in-vivo genotoxic response of IQC-γCD inclusion complex combining the comet assay on cells isolated from the liver of SD rats and micronucleus test in bone marrow cell tissues according to the OECD 489 and OECD 474 guidelines (OECD, 2016a, 2016b; Rothfuss et al., 2011). The in-vivo comet-micronucleus combined assay was chosen because of its ability to evaluate the genotoxic potential of xenobiotics metabolism. The xenobiotic-metabolizing system comprises several hundred enzymes, which are usually expressed with high selectivity in varying tissues, especially in the liver of mammals. The comet assay can determine the short-lived DNA damage, while the micronucleus assay detects the structural numerical chromosomal damage with precise sensitivity and specificity. Other advantages are the decrease in the number of false-negative results, and reduction in animal usage in the risk assessment process (Mughal et al., 2010; He et al., 2000; Kirkland et al., 2019) according to 3Rs principles (Replace, Reduce, and Refine) recommended by the OECD protocols (OECD, 2016b). Moreover, the OECD guidelines 474, recommended to use 5 animals per group and both sexes (OECD 1997d, 2020), but the current study was performed only with male SD rats. Hobbs et al. (2018) observed that alpha-glycosyl isoquercitrin induced more micronuclei in male mice than in females. Thus, to simplify the design of the present study, and according to ICH guideline S2 (R1) on genotoxicity testing and data interpretation for pharmaceuticals intended for human use (ICH, 2012), it was decided to conduct this study only in male SD rats, the highly sensitive sex to ensure the success of involved assays. Additionally, the OECD test guidelines for the micronucleus and comet assays were followed that describes the outline criteria for determination of a negative or positive response, based on statistical analysis (administrated groups compared to negative control; dose-response) comparison to laboratory historical data, and relevant tissue exposure. Based on the presented data, administration of 2000 mg/kg bw/day IQC-γCD inclusion complex did not produce DNA breaks in cells isolated from the liver of the SD rats in the comet assay. Also, the IQC-γCD inclusion complex exposure did not cause any statistically significant damage in the liver of SD rats. Therefore, we conclude the results of the comet assay in SD rats as negative. The absence of oxidative damage observed in DNA bases in-vivo could be related to either of two oxidative DNA repair pathways (Azqueta et al., 2009), an activation of nucleotide excision repair (NER) or base excision repair (BER).

Similarly, the negative result in the in-vivo rat bone marrow micronucleus assay reported here is consistent with previous reports in which soluble glycosides alpha-glycosyl isoquercitrin was administrated to both male and female rats (Hobbs et al., 2018; Salim et al., 2004). No significant changes in the PCE% incidence, MNPCE% incidence, and PCE/NCE ratios were observed in the SD rats exposed to the highest dose (2000 mg/kg bw/day) of IQC-γCD inclusion complex, and thus confirm a lack of bone marrow toxicity. This also suggests the absence of any simulated erythropoiesis that can lead to increases in MNPCE incidence as a consequence of a higher cell proliferation rate (Hayashi et al., 2007). A 13-week subchronic toxicity study with IQC-γCD inclusion complex exposure was recently conducted in Sprague Dawley (SD) rats, establishing no observable adverse effect levels (NOAEL) of 5.0% in the diet for male (3338.55 mg/kg bw/day) and 3.0% in the diet for female (2177.33 mg/kg bw/day) SD rats (Kapoor et al., 2021b)

In conclusion, in this study, it was demonstrated that the integration of both micronucleus and comet assays can be achieved by using a single study animal. Also, the study highlights the need for more consensus on how to define ambiguous Ames assay results that are not biologically relevant. Since the in-vivo comet assay can detect certain types of DNA damage very well (except induced crosslinking), the comet assay is an acceptable choice to detect potent genotoxins in the liver. Under the condition of the comet assay, no DNA damage was measured in the liver of SD rats exposed to different doses of IQC-γCD inclusion complex. Moreover, no induction of micronuclei was detected in response to administration of IQC-γCD inclusion complex in SD rats as no change in the MNPCE% incidence was noted indicating no bone marrow cytotoxicity at doses tested up to the limit dose of the assay. The results presented in this study are consistent with existing literature and indicating a complete lack of general in-vivo genotoxicity of flavonoids. In conclusion, the Ames test finding indicates that although there is some weakly positive in-vitro indication of genotoxic potential in the presence or absence of the metabolic activation system (S9 fraction from rat liver); however based on the no observable adverse effect levels (NOAEL) reported for IQC-γCD inclusion complex (Kapoor et al., 2021b), no biologically and toxicologically relevant mutagenicity at the conditions examined is recommended according to regulatory guidelines. Therefore, the unanimous opinion is that the comet and micronucleus assays in SD rats do not provide credible evidence of in-vivo genotoxic potential resulting from exposure to IQC-γCD inclusion complex, supporting its safe use as a functional food. Therefore, the comprehensive GLP-compliant safety assessment of the IQC-γCD inclusion complex described in this study indicates that the product showed a very mild mutagenesis /cytotoxicity but without any toxicological concerns. Thus, the IQC-γCD inclusion complex does not pose genotoxic concerns for humans at the presented estimated levels of dietary administration.

ACKNOWLEDGMENTS

Authors would like to thank the colleagues at BoZo Research Center Inc., Japan, and Taiyo Kagaku Co., Ltd., Japan for their kind support during this non-clinical study.

Conflict of interest

The authors have no competing interests and declare no potential conflict of interest. The product-related patent exists, but this does not alter the authors’ adherence to all the Journal of Toxicological Sciences policies on sharing data and materials. MPK, and MM are employees of Taiyo Kagaku, Japan. DT is an employee of Taiyo International, USA.

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